Alkaline phosphatase assay

ABSTRACT

Alkaline phosphatase is assayed in biological specimens by using granular solid, water-soluble substantially anhydrous, storagestable reagent formulations containing a nitrogen bearing polyoxyalkylene nonionic surfactant obtained by the sequential reaction of ethylenediamine with propylene oxide and ethylene oxide in the presence of a catalyst. The surfactant has an advantageous effect upon granulation, stabilization and dissolution of the formulations, and is effective for solubilizing proteins such as enzymes in the formulations or the specimen.

i United States Patent Monte et a1.

[ Dec. 16, 1975 ALKALINE PHOSPHATASE ASSAY Inventors: Alexander A.Monte; Ching Chiang,

both of Glendora, Calif.

Assignee: Mallinckrodt, Inc., St. Louis, Mo.

Filed: May 6, 1974 Appl. No.: 467,319

Related U.S. Application Data Division of Ser. No. 200,552, Nov. 19,1971, Pat. No.

2,816,262, which is a continuation-in-part of Ser. No. 190,883, Oct. 20,1971, abandoned.

U.S. Cl. 195/l03.5 R; 23/230 B; 195/99; 252/408 Int. Cl. COIN 31/14Field of Search 195/63, 68, 103.5 R, 100, 195/99; 23/230 B; 252/408References Cited UNlTED STATES PATENTS 9/1961 Babson 195/103.5 R

3,002,893 10/1961 Babson l95/103.5 R 3,413,198 11/1968 Deutsch 195/63 X3,425,912 2/1969 Deutsch et a1. 195/103.5 R 3,627,688 12/1971 McCarty eta1. 195/63 X Primary ExaminerDavid M. Naff Attorney, Agent, orFirm-Mathew D. Madsen [5 7 ABSTRACT 12 Claims, 1 Drawing Figure ALKALINEPI-IOSPHATASE ASSAY CROSS-REFERENCE TO RELATED APPLICATION Thisapplication is a division of our copending, coassigned US. patentapplication Ser. No. 200,552, filed Nov. 19, 1971, now US. Pat. No.2,816,262, which is a continuation-in-part of our US. patent applicationSer. No. 190,883, filed Oct. 20, 1971, now abandoned.

BACKGROUND OF THE INVENTION This invention relates to the field ofclinical diagnostic testing and more particularly to novel reagents andmethods for making biological assays on body fluids.

A large variety of test reagents and methods are available for use indetermining the character of various body fluids to assist in thediagnosis of certain pathological conditions. Tests for determination ofcertain types of biological activity or the presence and quantity ofcertain biologically active components provide information indicatingthe presence or absence of disease or other physiological disorder. Inaccordance with such tests, the biological specimen to be analyzed, forexample, a sample of a body fluid, is typically mixed with a liquidreagent formulation which contains a reagent capable of effecting areaction which causes a measurable change in the specimen/reagentsystem. Very often the reaction which takes place in the test is anenzymatic reaction. Certain tests are designed, in fact, to determinethe presence of a particular enzyme and in such cases the reagentformulation may contain a substrate upon which the enzyme to bedetermined is known to act. In other cases, the determination may be fora material which is known to be a reactive substrate in an enzymaticallycatalyzed reaction. In either case, the reagent formulation verycommonly contains an enzyme, a coenzyme or both. Because the catalyticactivity of most enzymes is specific to a particular reaction, testreagents can be formulated which are effective to determine specificbiological components or activities even in a complex body fluidcontaining a large number of other components which might interfere withefforts to obtain a purely chemical analysis. Moreover, many of thecomponents which are to be determined have highly complex chemicalstructures which would render direct chemical analysis difficult even inthe absence of any contaminants.

Unfortunately, enzymes and coenzymes are generally rather delicatematerials which may be readily denatured by heating and which also tendto degenerate upon storage. Many of the substrate materials used inbiological assay reagent formulations are similarly unstable. Liquidreagents containing such components are therefore not generallysusceptible to storage and must be freshly prepared shortly prior to usein clinical diagnostic testing. Because of the relative expense ofenzymes and coenzymes and the skill required to prepare a reagentformulation containing these materials which can be utilized to obtainaccurate clinical diagnostic test results, the instability of the liquidformulations has motivated a substantial amount of research to developreagents in a relatively storage-stable form. Much of this effort hasbeen directed to the develop ment of solid, dry, water-solubleformulations which can be dissolved in water at the time of testing toprovide a fresh liquid reagent useful in the test. Typical prior art dryreagent formulations are disclosed in 2 Deutsch US. Pat. No. 3,4l3,l98and Stern et al. US. Pat. No. 3,546,131.

A dry reagent formulation satisfactory for use in preparing liquidreagents for routine clinical diagnostic tests should satisfy a numberof criteria. It must be readily soluble in a solvent compatible with thebiological specimen, usually water. It should be capable of solubilizingproteinaceous material in the specimen. Moreover, it should be readilysusceptible to packaging in convenient sized packages and be adapted forrapid dissolution in the solvent to provide a liquid reagent of properstrength for a given test or series of tests.

SUMMARY OF THE INVENTION It is an object of the present invention toprovide improved dry, water-soluble, reagent formulations for use inconducting clinical diagnostic tests. It is a further object of thepresent invention to provide such formulations which can be readilygranulated and shipped or stored in granular form. It is a particularobject of the invention to provide such reagent formulations infreeflowing, granular form at consistent bulk densities so that they maybe delivered to a volumetric packaging or tableting operation inconsistent weight amounts. Additional objects of the invention includethe provision of dry reagent formulations having a high capacity forsolubilizing protein; the provision of such formulations having a highdegree of storage stability; the provision of methods for preparing thedry reagent formulations of the invention; and the provision of methodsfor conducting clinical diagnostic tests utilizing such reagentformulations. Other objects and features will be in part apparent and inpart pointed out hereinafter.

In one of its aspects, therefore, the present invention is directed to areagent formulation for use in conducting a clinical diagnostic test ona biological specimen. The reagent formulation comprises a solid,water-soluble, substantially anhydrous, storage-stable mixturecontaining a reagent capable of participating in a test reaction toeffect a measurable change in a test system, and a solidnitrogen-containing polyoxyalkylene nonionic surfactant. The surfactanthas a structure corresponding to that obtained when ethylene diamine isreacted sequentially with propylene oxide and ethylene oxide in thepresence of a catalyst and the polyoxypropylene chains of the surfactanthave an average molecular weight of between about 750 and about 6750.

The invention is further directed to a method of conducting a clinicaldiagnostic test on a biological specimen using the aforementionedreagent formulation. The method comprises dissolving the reagentformulation in water to produce a liquid reagent; mixing the liquidreagent with a specimen to form a specimen/reagent test system; andmeasuring a change in the system resulting from the reaction between thereagent and the specimen.

The invention is also directed to a method of preparing the novelreagent formulation. The method com prises the steps of mixing a reagentcapable of partici pating in a test reaction to effect a measurablechange in a test system, a nitrogen-containing polyexyalkylene nonionicsurfactant of the above-noted character, and a solvent for thesurfactant; and removing the solvent to form a substantially anhydrous,water-soluble, freeflowing, granular solid.

DESCRIPTION OF THE DRAWING The drawing is a grid illustrating themolecular structure of various commercially available nonionicsurfactants useful in the practice of the invention. The coordinates ofeach point on the grid correspond to the chain size of thepolyoxyethylene hydrophile and poly oxypropylene hydrophobe moieties ofa particular surfactant. Boundary lines set out on the grid separate theareas encompassing surfactants which assume different physical states.

DESCRIPTION OF THE PREFERRED EMBODIMENTS To facilitate preparation ofliquid reagents from solid formulations in the clinical laboratory, itis highly desirable to package the solid formulations in proper unitaryamounts. Thus, for example, the solid formulations may be encapsulatedor tabletted with the proper quantity of reagent in each capsule ortablet for conducting a single test. Alternatively, a multitest packagecan be provided from which the proper amount of liquid reagent isprepared for conducting a specified number of tests.

Where a solid reagent formulation is packaged in unitary amounts,accuracy of metering the solid material into each capsule, tablet ormulti-test package is important. The metering equipment which is usedfor delivering solid materials in packaging and tableting operations,however, almost universally operates on a volumetric basis. Unless thesolid material is free-flowing and has a consistent bulk density,therefore, it cannot be delivered in consistent weight amounts to eachpackage, capsule or tableting station using conven tional equipment.

To provide a solid formulation in free-flowing form of consistent bulkdensity, it is preferably granulated prior to packaging. Granulationconverts a powdered material into a material constituted by smallagglomerates of relatively uniform size. Properly prepared, the granularmaterial is free-flowing, has a consistent bulk density and is readilyhandled by the metering devices used in packaging operations. Togranulate a powdered material, the powder is typically mixed with abinder dissolved in a volatile solvent, wet screened, dried by drivingoff the solvent, and dry screened following the drying step. In additionto the binder, a lubricating substance is normally incorporated in thegranulation mass to further enhance the flow characteristics of thegranules, especially under the compressive stress of tabletingoperations.

As noted, solid formulations useful as reagents for conducting clinicaldiagnostic tests on biological speci mens should have certain additionalproperties. Because they are dissolved in water to produce a liquidreagent, all components, including the binder, should be readilywater-soluble. Because many of the tests involve enzymatic reactionsand/or proteinaceous substrates, the formulation should possessdetergent properties for solubilizing protein.

It has now been discovered that the above objectives can be met and thateffective clinical reagent formulations for the determination of certainbiological properties of body fluids can be produced in free-flowing,granular form through the use of particular nitrogenbearingpolyoxyalkylene nonionic surfactants. Test formulations granulated withthe aid of these surfactants are well adapted to precision packaging andtableting operations. Because of their free-flowing character andconsistent bulk density, they can be delivered to either a packaging ortableting operation in consistent weight amounts by volumetric metering.As a consequence, clinical test reagents formulated at a centrallocation remote from a clinical laboratory can be utilized to prepareliquid test reagents for clinical use without the need for weighing,analyzing, or other procedures by the clinical chemist or technician.

The nitrogen-containing surfactants which are useful in the formulationsof the invention possess the unique multiple capability of serving asbinders, lubricants and solubilizers for protein. Moreover, they arethemselves water-soluble, thus promoting the dissolution of the reagentformulations in water to provide clinical liquid reagents. Thesesurfactants are sold under the trade designation Tetronic by WyandotteChemical Corporation. They are normally prepared by sequential reactionof first propylene oxide and then ethylene oxide with ethylene diaminein the presence of an alkaline or acid catalyst. Normally thesesurfactants are prepared at elevated temperatures using alkalinecatalysts such as sodium hydroxide, potassium hydroxide, sodiumalkoxide, quarternary ammonium bases and the like. Other methods areavailable for the preparation of these surfactants. The preparation ofsurfactants such as those utilized in the formulations of the inventionis more fully described in US. Pat. No. 2,979,528.

The properties and physical state of nonionic surfac tants havingstructures corresponding to those derived from ethylene diamine,propylene oxide and ethylene oxide vary with the lengths of thepolyoxypropylene and polyoxyethylene chains. As the drawing shows, thephysical state of these surfactants is largely dependent upon theproportionate weight of the surfactant constituted by thepolyoxyethylene chains, but is also influenced by the average molecularweight of the polyoxypropylene moieties. The polyoxypropylene chains arehydrophobic while the polyoxyethylene chains are hydrophilic. Thus, thesurfactants having polyoxypropylene units of low average molecularweight are more watersoluble than those having polyoxypropylene units ofa higher average molecular weight. The numbers set out on the face ofthe grid correspond to particular members of the Tetronic series. Eachnumber is located at a point on the grid whose coordinates correspond tothe polyoxyethylene and polyoxypropylene chain sizes of the particularproduct which is commercially designated by said number.

Essentially any surfactant whose structure is defined by the coordinantsof a point lying in the grid of 'the drawing may be utilized in theformulations of the invention. It is preferred, however, that thesurfactant be solid or at least semi-solid. A greater proportion of thesolid surfactants can be satisfactorily incorporated in a reagentformulation and thus a greater binding and lubricating capacity isobtained without adversely affecting other properties of theformulation. Desirably, on the order of 2.5 to 5% by weight of thepreferred solid surfactants are incorporated in the reagentformulations. When the liquid formulations are used, it is not alwayspossible to incorporate more than 2 or 3% by weight of the surfactantwithout imparting a somewhat waxy character to the formulation. The useof 2 to 3% by weight of a liquid Tetronic surfactant produces a usefulproduct,but the binding and lubricating capabilities of the surfactantare not always fully exploited at such a level. Granules having the mostdesirable properties are obtained using solid or semi-solid surfactants.

Since the dry reagent formulations of the invention are dissolved inwater for use in conducting clinical diagnostic tests, it is alsodesirable that the surfactant component promote the dissolution of thegranular product. Thus, it is preferred that the surfactant be ashydrophilic as possible, i.e., that the molecular weight of thepolyoxypropylene hydrophobe moiety of the surfactant be relatively low.Thus, the preferred surfactants for use in the formulations of theinvention are those which are both solid or semi-solid in physical stateand relatively hydrophilic. Solid-state surfactants withpolyoxypropylene chains having an average molecular weight of less thanabout 4000 are especially preferred, with the most suitable surfactantsbeing those whose polyoxypropylene chains have an average molecularweight of between about 2750 and about 3750 and whose weight percentageof polyoxyethylene units is between about 70% and about 80%. Two particular surfactants whose weight and structure characteristics fallwithin the latter limits are those sold under the trade designationsTetronic 707 and Tetronic 908". Tetronic 707 has a polyoxypropylenehydrophobe molecular weight on the order of 2750 and a weight percentageof polyoxyethylene units of about 70 while Tetronic 908" has apolyoxypropylene molecular weight of about 3750 and a weight percentageof polyoxyethylene units of about 80%. Good results are also obtainedwith surfactants whose polyoxypropylene chains have an average molecularweight of between 750 and 4000 with a weight percentage of between about35% and about 65% polyoxyethylene units. Other surfactants within thegrid of the drawing are reasonably satisfactory but less effective thanthose represented by the right lower corner of the grid.

In addition to their advantageous effect upon granulation anddissolution of dry clinical test reagent formulations, surfactants ofthe above-noted character have been found to be effective forsolubilizing protein. As indicated above, this is a highly advantageouscharac teristic, since enzymes and other proteinaceous matter derivedfrom either the reagent formulation or the specimen commonly participatein the test reactions. By solubilizing protein, the surfactants functionto facilitate the progress of the test reaction and thus enhance theeffectiveness of the reagent formulation. It may, therefore, be seenthat incorporation of these surfactants in clinical test formulationsuniquely provides multiple advantages in the preparation, packaging,dissolution and functional operation of clinical reagent formulations.

It has further been discovered that the dry clinical reagentformulations of the invention are quite stable and generally possessgood shelf life characteristics. Although we cannot precisely accountfor the particular ingredient or combination of ingredients whichimparts the high degree of storage stability, it appears that suchstability may be a somewhat general characteristic of dry clinicalreagent formulations which include the particular nitrogen-containingnonionic surfactants used in our formulations. If so, the ability toimpart storage-stability represents a further aspect of the uniquemultiple function of this type of surfactant in such formulations.

To prepare the reagent formulations of the invention, the surfactant ismixed with a volatile solvent and at least one reagent capable ofparticipating in a test reaction to effect a measurable change in areagent/specimen test system. The surfactant should be soluble up to theamount present in the solvent which is utilized. Solvents which may beused include methylene chloride, chloroform, methanol, benzene, water,methanol/water, and chloroform/methylene chloride. After thorough mixingand appropriate size classification, the solvent is removed to yield agranular product.

In a preferred embodiment of the invention, the ingredients of theformulation, in dry particulate form, are thoroughly blended in amechanical mixer. With the mixer running, a granulating solutioncontaining the solvent and the surfactant, preferably that sold underthe trade designation Tetronic 707 or Tetronic 908, is added. Additionalsolvent is used as needed to produce granular agglomerates of thedesired size and wetness.

The resulting wet granulation is screened through a coarse screen, forexample 10 mesh, then spread in thin layers in trays and dried atreduced pressure, for exam ple, 25 inches Hg absolute or less. Dependingon the heat sensitivity of the formulation, drying is normally carriedout at room temperature or at modest elevated temperature (up to about37C.). Generally, the depth of the wet granules in the trays should notexceed about /2 inch to inch.

After completion of the drying cycle, the dried granulation isrescreened through a finer screen, for example, 20 to 30 mesh, blendedthoroughly and packaged in containers essentially impervious tomoisture. Since the components of the reagent formulation are frequentlymoisture sensitive, the formulation should not be exposed to a relativehumidity of more than about 5% after removal from the dryer.

The reagent formulations of the invention are adapted to be packaged insmall unitary packages. For example, sufficient reagent formulation fora single assay may be tabletted or packaged in a capsule. The reagentformulations are also adapted to packaging in such containers as foilstrip packets, utilizing automatic packaging machinery. Utilizing thispackaging approach, sufficient reagent formulation to carry out asuitable predetermined number of tests, such as 10, 25, or 50 tests, maybe accurately packaged in a single foil packet. The user then simplydissolves the contents of the multiple test packet in a predeterminedvolume of water and uses a suitable aliquot of the resulting liquidreagent in the performance of each of a series of assays for the desiredbiological substance or property.

In some instances, depending on the nature of the components and theircompatibility, all of the reagents necessary in a single assay ordetermination may be included in a single formulation. In otherinstances, incompatabilities and/or other considerations may make itdesirable to segregate certain reagents in which case two or morereagent formulations are prepared in accordance with the invention.

To conduct a clinical diagnostic test using the formulations of theinvention, the liquid reagent produced by dissolving the dry formulationin a pre-determined amount of water is mixed with the biologicalspecimen in a predetermined volumetric or weight ratio. With the aid ofappropriate instrumentation as required, the resulting specimen/reagentsystem is observed for the presence, absence, nature and extent of aphysical, chemical or biological change. Such change as does occur ismeasured to provide the desired information for use in the clinicaldiagnosis.

Exemplary reagent formulations prepared in accordance with the inventionand useful for the determination of hemoglobin, blood urea nitrogen,total protein, serum glutamic oxaloacetic transaminase, alkalinephosphatase, glucose, inorganic phosphorus, lactate dehydrogenase-L,serum glutamic pyruvic transaminase, uric acid (colorimetric) and uricacid (u.v.) are set forth in Table l. The preferred compositions ofthese reagent formulations and methods for preparing them are describedin the examples following Table l which more fully illustrate theinvention.

TABLE 1 Exemplary Clinical Test Reagent Formulations Dry IngredientsFormu- Type of Formu (Reagents. Etc.)

Granulating Solution Poly No. of ethylene Theo- Tests TETRONIC glycolCH. ,Cl retical tThou lation lation Name/Formula WT. (g) 707 (g.) 6000(g.) (ml) (1) Yield (g.) sands) A Reagent Formu NaHCO; 300 30 (a) 200I000 50 lation for Hemo- K;,Fe(CN). 50 (b) 300 glohin Assay KCN 30Mannitol 590 H Reducer Formu Ascorbic Acid 3250 l50 (a) 500 5000 50lation for Alkaline Sulfamic Acid 1600 (b) 200 Phosphatase Assay lMolybdate Formu Sodium Molyh- 475 37.5 (a) 300 l250 50 lation forAlkaline date 27(H O) Phosphatase Assay Duponol ME Dry 737.5 (b) 150 JSubstrate Formu Disodium B-Glycerolation for Alkaphosphate 500 87.5 (a)400 2600 50 line Phosphatase Tris-(hydroxymethyU- Assay aminomethane1500 100 Succinic Acid 125 Duponol ME Dry 500 t l (:1) indicates amountof CH Cl used as carrier for Tetronic 707. (h) indicates amount ofadditional CH- .Cl. used to optimize granulation.

EXAMPLE 1 Hemoglobin Reagent Formulation and Assay Composition of thereagent formulation useful for hemoglobin assay is set forth asformulation A in Table 1.

To prepare this formulation, sodium bicarbonate (300 g.). milledpotassium ferricyanide (50 g.) and potassium cyanide (30 g.) wereinitially added to a Hobart bowl and mixed with a stainless steelspatula. Mannitol (590 g.) was then added and the resulting blend wasagitated for 5 minutes in the mixer. While agitation was continued, asolution of Tetronic 707 (30 g.) in methylene chloride (200 ml.) wasadded. An additional amount of methylene chloride (300 ml.) was thenadded to produce the proper granulation.

The wet granulation was screened through a No. mesh stainless steelscreen and the wet screened material was transferred to 8 inch X 12 inchPyrex drying trays, at a depth of between about /2 inch and about inchesin each tray. The granulation was then dried in a vacuum oven for 15hours at a temperature of C. and a pressure of 25 inches Hg.

The dried granulation was removed from the vacuum oven in an environmentwhere the relative humidity was not more than 5%. The dried granulationwas then screened through a No. 20 mesh stainless steel screen using anErweka oscillator. The screened, dried granulation was transferred to aP. K. blender and mixed for 5 minutes, then packaged in tightly closedcontainers. Approximately 1000 g. of a water-soluble, substantiallyanhydrous reagent formulation, sufficient for 50,000 tests, wasobtained.

LII

sity of the reagent/specimen system is measures at 540 nm. using asuitable spectrophotometer and compared against a reagent blank set at100% transmission. The hemoglobin level is then determined by referenceto a standard curve.

To prepare a liquid reagent sufficient for tests, formulation A (1.00g.) is dissolved in distilled water and the resulting solution isdiluted to 250 ml. and mixed thoroughly. The reagent solutionthusproduced is stable for three months at room temperature if protectedfrom light.

To conduct the hemoglobin assay test, a reagent/- specimen test systemis prepared by adding 20 microliters of well mixed blood (collected withan anticoagulant) to 5 ml. of the above solution of formulation A in aclean test tube. The contents of the tube are mixed thoroughly andallowed to stand at room temperature for at least 5 minutes. The opticaldensity is then measured as described above to determine the hemoglobinlevel.

EXAMPLE 2 Alkaline Phosphatase Formulations and Assay Three separateformulations are provided for the alkaline phosphatase test. Theseformulations are set forth in Table l as formulations H. I and J.Predetermined amounts of these formulations dissolved in separateportions of water provide liquid reagents for use in making the alkalinephosphatase assay.

Prior to the preparation of chromogenic reagent formulation I, thesodium molybdate component of the reagent blend was dried. Sodiummolybdate was transferred into tared 8 inch X 12 inch Pyrex trays at adepth of between about /2 inch and /1 inch. The trays were 9 then placedin a vacuum oven and the sodium molybdate dried at 55C. and a totalpressure of inches Hg until a weight loss of not less than 13% wasobserved.

To prepare chromogenic reagent formulation 1, the dried sodium molybdate(475 g.) and a dry sodium lauryl sulfate product sold under the tradedesignation Duponol ME by E. l. DuPont de Nemours and Company (737.5 g.)were blended in a Hobart bowl and agitated to promote intimate mixing.With the mixer running. a solution of Tetronic 707 (37.5 g.) inmethylene chloride (300 ml.) was introduced. Additional methylenechloride 150 ml.) was subsequently added to produce the desired degreeof granulation and wetness. The wet granulation was screened and driedand the resultant dry granulation rescreened and packaged in the mannerdescribed in Example 1 for hemoglobin reagent formulation A.

In the preparation of reducer quench formulation H, L-ascorbic acid 3.25kg.) and sulfamic acid 1.60 kg.) were blended in a Hobart bowl andagitated to promote intimate mixing. With the mixer running, a solutionof Tetronic 707 (150 g.) in methylene chloride (500 ml.) was introduced.Additional methylene chloride (200 ml.) was subsequently added toproduce the desired degree of granulation and wetness. The wetgranulation was screened and dried and the resultant dry granulationrescreened and packaged in the manner described in Example 1 forhemoglobin reagent formulation A.

Preparation of buffer substrate reagent formulation J was initiated byblending B-glycerophospheric acid, disodium salt (500 g.)tris-(hydroxymethyl)-aminomethane (1.5 kg.), milled succinic acid (12.5g.) and Duponol ME" (500 g.) in a Hobart bowl and agitating the blend topromote intimate mixing. With the mixer running. a solution of Tetronic707" (87.5 g.) in methylene chloride (400 ml.) was introduced.Additional methylene chloride (100 ml.) was subsequently added toproduce the desired degree of granulation and wetness. The wetgranulation was screened and dried and the resultant dry granulationrescreened and packed in the manner described in Example 1 forhemoglobin reagent formulation A.

Dissolved in separate portions of water, formulations H, I and J provideliquid reagents useful in the determination of the alkaline phosphatasecontent of biological specimens such as blood serum. Alkalinephosphatase in the specimen promotes the release of phosphate from the,B-glycerophosphate constituent of substrate reagent formulation J. Thephosphate thus released reacts with sodium molybdate of chromogenicreagent formulation I in the presence of sulfamic acid and as corbicacid of reducer quench formulation H. The ascorbic acid reduces thephosphomolybdic acid thus formed to phosphomolybdenum blue. The color ofthe latter is indicative of the concentration of alkaline phosphatasepresent in the reagent specimen system.

In preparation for a test, the dry reagent formulations are eachdissolved in water to provide liquid reagents. Formulation I 1.25 g.) isdissolved in 25 ml. of distilled water to provide a chromogenic liquidreagent; formulation H (5.00 g.) is dissolved in 25 ml. of water toprovide a reducer quench liquid reagent: and formulation J (2.60 g.) isdissolved in 150 m1. of water to provide a substrate buffer liquidreagent. The solution of formulation I is stable for a week at roomtemperature. The solution of formulation J is stable for two weeks whenrefrigerated. The liquid reagent constituted by 10 the solution offormulation H should be prepared fresh daily and protected from light.The resulting solutions are sufficient for conducting 50 tests.

Conduct of an alkaline phosphatase determination is initiated by adding3 ml. portions of the solution of substrate formulation J to each of twotest tubes and incubating each tube for 2 minutes at 37C. One of the twotubes is then used for the test reaction while the other tube is usedfor a blank.

0.1 ml. lambda) of serum is added to the tube used for the test reactionand mixed thoroughly with the liquid reagent therein to provide aspecimen/reagent test system. The test system is then incubated forexactly 15 minutes at 37C., following which 0.5 ml. portions of theliquid reagent solution of formulation I and 0.5 ml. portions of theliquid reagent solution of formulation H are added to both the testsystem and the tube carrying the test blank. Both the test system andthe blank are then incubated at 37C. for another 20 minutes, and theoptical density of each determined at 700 nm against a water blank setat 100% transmission. Using a standard curve, the inorganic phosphorouscontent of each test solution is determined from its optical densitycompared to the water blank. Since both the test system and test blankoriginally contain the same amount or proportion of B-glycerophosphate,the inorganic phosphorous released by action of alkaline phosphataseisdetermined by subtracting the total inorganic phosphorous in the testsystem from the total amount of inorganic phosphorus in the blank.

To calculate the'alkaline phosphatase units present in the test system,the mg% inorganic phosphorus released is multiplied by 4. In theprocedure of Bodansky, known to the art, there is a one-to-onecorrespondence between the mg% phosphorus released and the alkalinephosphatase units. Since the test of the invention utilizes only a 15minute incubation time instead of the 60 minute incubation time ofBodansky, the factor of 4 is applied to obtain corresponding results.

The standard curve used to determine absolute inorganic phosphorus isobtained by spectrophotometric measurements of the optical densities ofpotassium dihydrogen phosphate solutions containing 0, 2.5, 5.0. 7.5 and10 mg% phosphorus at 700 nm, against a reagent blank set at 100%transmission. A standard potassium dihydrogen phosphate solution for usein establishing the standardcurve is prepared by weighing out KH- PO(438.1 mg.) in a 100 ml. volumetric flask. diluting to volume with highquality distilled water, and storing the diluted solution at 4C. Thisstock solution contains 100 mg. phosphorus per 100 m1. (100 mg%) andappropriately diluted aliquots of this stock solution are used inestablishing the standard curve.

As those skilled in the art will appreciate, phosphorus standard curvesobtained from spectrophotometric measurements of varying concentrationsof potassium dihydrogen phosphate are linear only up to about 15 mg%.Where high phosphorus sera (above 7.5 mg%) are analyzed for alkalinephosphatase content. therefore. a 50 lambda serum sample is used and thefinal result multiplied by 2.

An alkaline phosphatase unit is defined as the amount of enzyme in 100ml. of serum which releases 1 mg. phosphorus per hour at 37C.

In view of the above. it will be seen that the several objects of theinvention are achieved and other advantageous results attained.

As various changes could be made in the above methods and productswithout departing from the scope of the invention, it is intended thatall matter contained in the above description shall be interpreted asillustrative and not in a limiting sense.

What is claimed is:

1. A buffer substrate reagent formulation for usein assaying abiological specimen for alkaline phosphatase comprising a solid.water-soluble. substantially anhydrous. storage-stable mixturecontaining:

' a. B-glycerophosphoric acid disodium salt:

b. tris-( hydroxymethyl kaminomethane;

c. succinic acid:

d. sodium lauryl sulfate; and

e. a nitrogen-containing polyoxyalkylene nonionic surfactant obtained bythe sequential reaction of ethylenediamine with propylene oxide andethylene oxide in the presence of a catalyst. said surfactant containingpolyoxypropylene chains having an average molecular weight of betweenabout 750 and about 6750 and polyoxyethylene chains constituting betweenabout 10 and about 80 weight percent of said surfactant.

2. A reagent formulation as set forth in claim 1 wherein saidnitrogen-containing surfactant is solid and the polyoxypropylene chainsthereof have an average molecular weight of less than about 4000.

3. A reducer quench reagent formulation for use in assaying a biologicalspecimen for alkaline phosphatase comprising a solid. water-solublesubstantially anhydrous. storage-stable mixture containing:

a. L-ascorbic acid:

b. sulfamic acid; and

c. a nitrogen-containing polyoxyalkylene nonionic surfactant obtained bythe sequential reaction of ethylenediamine with propylene oxide andethylene oxide in the presence of a catalyst. said surfactant containingpolyoxypropylene chains having an average molecular weight of betweenabout 750 and about 6750 and polyoxyethylene chains constituting betweenabout 10 and about 80 weight percent of said surfactant.

4. A reagent formulation as set forth in claim 3 wherein saidnitrogen-containing surfactant is solid and the' polyoxypropylene chainsthereof have an average molecular weight of less than about 4000. 5. Achromogenic reagent formulation for use in assaying a biologicalspecimen for alkaline phosphatase comprising a solid. water-solublesubstantially anhydrous. storage-stable mixture containing:

a. sodium molybdate;

b. sodium lauryl sulfate; and i c. a nitrogen-containing polyoxyalkylenenonionic surfactant obtained by the sequential reaction of ethylenediamine with propylene oxide and ethylene oxide in the presence of acatalyst said surfactant containing polyoxypropylene chains having anaverage molecular weight of between about 750 and about 6750 andpolyoxyethylene chains constituting between about 10 and about 80 weightpercent of said surfactant.

6. A reagent formulation as set forth in claim 5 wherein saidnitrogen-containing surfactant is solid and the polyoxypropylene chainsthereof have an average molecular weight of less than about 4000.

7. The method of assaying a biological specimen for alkaline phosphataseusing a plurality of solid. water- 12 soluble. substantially anhydrous.storage-stable reagent formulations respectively comprising:

1. a chromogenic reagent formulation containing:

a. sodium molybdate;

b. sodium lauryl sulfate; and

c. a nitrogen-containing polyoxyalkylene nonionic surfactant obtained bythe sequential reaction of ethylenediamine with propylene oxide andethylene oxide in the presence of a catalyst, said surfactant containingpolyoxypropylene chains having an average molecular weight of betweenabout 750 and about 6750 and polyoxyethylene chains constituting betweenabout 10 and about weight percent of said surfactant;

2. a reducer quench reagent formulation containing:

a. L-ascorbic acid; b. sulfamic acid; and c. said nitrogen-containingpolyoxyalkylene nonionic surfactant; and 3. a buffer substrate reagentformulation containing:

a. B-glycerophosphoric acid disodium salt; b. tris-( hydroxymethyl)-aminomethane: c. succinic acid; d. sodium lauryl sulfate; and e. saidnitrogen-containing polyoxyalkylene nonionic surfactant;

the method comprising the steps of:

i. dissolving each of said reagent formulations in separate portions ofwater to produce a plurality of liquid reagents;

ii. preheating an aliquot of predetermined volume of the liquid reagentcontaining said buffer substrate reagent formulation for a predeterminedperiod'of time;

iii. mixing the preheated aliquot of said liquid reagent containing saidbuffer substrate reagent formulation with a biological specimen to forma specimen/reagent test system;

iv. maintaining the specimen/reagent test system at a predeterminedelevated temperature for a predetermined period of time;

v. adding both the liquid reagent containing said chromogenic reagentformulation and the liquid reagent containing said reduced quenchreagent formulation to said system;

vi. thereafter maintaining said system at a predetermined elevatedtemperature for a further predetermined period of time: and

vii. measuring the optical density of said system resulting from theinteraction between said reagents and said specimen.

8. The method as set forth in claim 7 wherein the optical density ofsaid system is compared with the optical density of a blank processedunder such conditions as to provide the same proportion of inorganicphosphorus as would be provided in said test system in the absence ofalkaline phosphatase.

9. The method as set forth in claim 7 wherein said nitrogen-containingpolyoxyalkylene surfactant is solid and the average molecular weight ofthe polyoxypropylene chains thereof is less than about 4000.

10. The method of preparing a solid. water-soluble. free-flowing.substantially anhydrous. storage-stable chromogenic reagent formulationfor use in assaying a biological specimen for' alkaline phosphatase.said method comprising the steps of:

l. prreparing a mixture containing:

a. sodium molybdate;

b. sodium lauryl sulfate;

c. a nitrogen-containing polyoxyalkylene nonionic surfactant obtained bythe sequential reaction of ethylenediamine with propylene oxide andethylene oxide in the presence of a catalyst, said surfactant,containing polyoxypropylene chains hav ing an average molecular weightof between about 750 and about 6750 and polyoxyethylene chainsconstituting between about and about 80 weight percent of saidsurfactant; and

d. a solvent for said surfactant; and

2. removing the solvent to form a substantially anhy drous,free-flowing, water-soluble, granular solid.

11. The method of preparing a solid, water-soluble, free-flowing,substantially anhydrous, storage-stable buffer substrate reagentformulation for use in assaying a biological specimen for alkalinephosphatase, said method comprising the steps of:

1. preparing a mixture containing:

a. fi-glycerophosphoric acid disodium salt;

bi tris-( hydroxymethyl )-aminomethane;

c. succinic acid;

d. sodium lauryl sulfate;

e. a nitrogen-containing polyoxyalkylene nonionic surfactant obtained bythe sequential reaction of ethylenediamine with propylene oxide andethylene oxide in the presence of a catalyst, said surfactant containingpolyoxypropylene chains hav- U reducer quench reagent formulation foruse in assaying a biological specimen for alkaline phosphatase, saidmethod comprising the steps of:

l. preparing a mixture containing:

a. L-ascorbi'c acid;

b. sulfamic acid;

c. a nitrogen-containing polyoxyalkylene nonionic surfactant obtained bythe sequential reaction of ethylenediamine with propylene oxide andethylene oxide in the presence of a catalyst. said surfactant,containing polyoxypropylene chains having an average molecular weight ofbetween about 750 and about 6750 and polyoxyethylene chains constitutingbetween about 10 and about weight percent of said surfactant; and

d. a solvent for said surfactant; and

2. removing the solvent to form a substantially anhydrous, free-flowing,water-soluble, granular solid.

l l i

1. A CHROMOGENIC REAGENT FORMULATION CONTAINING: A. SODIUM MOLYBDATE; B.SODIUM LAURYL SULFATE; AND C. A NITROGEN-CONTAINING POLYOXYALKYLNONIONIC SURFACTANT OBTAINED BY THE SEQUENTIAL REACTION OFETHYLENEDIAMINE WITH PROPYLENE OXIDE AND ETHYLENE OXIDE IN THE PRESENCEOF A CATALYST, SAID SURFACTANT CONTAINING POLYOXYPROPYLENE CHAINS HAVINGAN AVERAGE MOLECULAR WEIGHT OF BETWEEN ABOUT 750 AND ABOUT 6750 ANDPOLYOXYETHYLENE CHAINS CONSTITUTING BETWEEN ABOUT 10 AND ABOUT 80 WEIGHTPERCENT OF SAID SURFACTANT;
 1. preparing a mixture containing: a.L-ascorbic acid; b. sulfamic acid; c. a nitrogen-containingpolyoxyalkylene nonionic surfactant obtained by the sequential reactionof ethylenediamine with propylene oxide and ethylene oxide in thepresence of a catalyst, said surfactant, containing polyoxypropylenechains having an average molecular weight of between about 750 and about6750 and polyoxyethylene chains constituting between about 10 and about80 weight percent of said surfactant; and d. a solvent for saidsurfactant; and
 1. preparing a mixture containing: a. Beta-glycerophosphoric acid disodium salt; b.tris-(hydroxymethyl)-aminomethane; c. succinic acid; d. sodium laurylsulfate; e. a nitrogen-containing polyoxyalkylene nonionic surfactantobtained by the sequential reaction of ethylenediamine with propyleneoxide and ethylene oxide in the presence of a catalyst, said surfactantcontaining polyoxypropylene chains having an average molecular weight ofbetween about 750 and about 6750 and polyoxyethylene chains constitutingbetween about 10 and about 80 weight percent of said surfactant; and f.a solvent for said surfactant; and
 1. prreparing a mixture containing:a. sodium molybdate; b. sodium lauryl sulfate; c. a nitrogen-containingpolyoxyalkylene nonionic surfactant obtained by the sequential reactionof ethylenediamine with propylene oxide and ethylene oxide in thepresence of a catalyst, said surfactant, containing polyoxypropylenechains having an average molecular weight of between about 750 and about6750 and polyoxyethylene chains constituting between about 10 and about80 weight percent of said surfactant; and d. a solvent for saidsurfactant; and
 1. a chromogenic reagent formulation containing: a.sodium molybdate; b. sodium lauryl sulfate; and c. a nitrogen-containingpolyoxyalkylene nonionic surfactant obtained by the sequential reactionof ethylenediamine with propylene oxide and ethylene oxide in thepresence of a catalyst, said surfactant containing polyoxypropylenechains having an average molecular weight of between about 750 and about6750 and polyoxyethylene chains constituting between about 10 and about80 weight percent of said surfactant;
 1. A buffer substrate reagentformulation for use in assaying a biological specimen for alkalinephosphatase comprising a solid, water-soluble, substantially anhydrous,storage-stable mixture containing: a. Beta -glycerophosphoric aciddisodium salt; b. tris-(hydroxymethyl)-aminomethane; c. succinic acid;d. sodium lauryl sulfate; and e. a nitrogen-containing polyoxyalkylenenonionic surfactant obtained by the sequential reaction ofethylenediamine with propylene oxide and ethylene oxide in the presenceof a catalyst, said surfactant containing polyoxypropylene chains havingan average molecular weight of between about 750 and about 6750 andpolyoxyethylene chains constituting between about 10 and about 80 weightpercent of said surfactant.
 2. A reagent formulation as set forth inclaim 1 wherein said nitrogen-containing surfactant is solid and thepolyoxypropylene chains thereof have an average molecular weight of lessthan about
 4000. 2. a reducer quench reagent formulation containing: A.L-ascorbic acid; b. sulfamic acid; and c. said nitrogen-containingpolyoxyalkylene nonionic surfactant; and
 2. removing the solvent to forma substantially anhydrous, free-flowing, water-soluble, granular solid.2. removing the solvent to form a substantially anhydrous, free-flowing,water-soluble, granular solid.
 2. A REDUCER QUENCH REAGENT FORMULATIONCONTAINING: A. L-ASCORBIC ACID; B. SULFAMIC ACID; AND C. SAIDNITROGEN-CONTAINING POLYOXYALKYLENE NONIONIC SURFACTANT; AND
 2. removingthe solvent to form a substantially anhydrous, free-flowing,water-soluble, granular solid.
 3. a buffer substrate reagent formulationcontaining: a. Beta -glycerophosphoric acid disodium salt; b.tris-(hydroxymethyl)-aminomethane; c. succinic acid; d. sodium laurylsulfate; and e. said nitrogen-containing polyoxyalkylene nonionicsurfactant; the method comprising the steps of: i. dissolving each ofsaid reagent formulations in separate portions of water to produce aplurality of liquid reagents; ii. preheating an aliquot of predeterminedvolume of the liquid reagent containing said buffer substrate reagentformulation for a predetermined period of time; iii. mixing thepreheated aliquot of said liquid reagent containing said buffersubstrate reagent formulation with a biological specimen to form aspecimen/reagent test system; iv. maintaining the specimen/reagent testsystem at a predetermined elevated temperature for a predeterminedperiod of time; v. adding both the liquid reagent containing saidchromogenic reagent formulation and the liquid reagent containing saidreduced quench reagent formulation to said system; vi. thereaftermaintaining said system at a predetermined elevated temperature for afurther predetermined period of time; and vii. measuring the opticaldensity of said system resulting from the interaction between saidreagents and said specimen.
 3. A reducer quench reagent formulation foruse in assaying a biological specimen for alkaline phosphatasecomprising a solid, water-soluble substantially anhydrous,storage-stable mixture containing: a. L-ascorbic acid; b. sulfamic acid;and c. a nitrogen-containing polyoxyalkylene nonionic surfactantobtained by the sequential reaction of ethylenediamine with propyleneoxide and ethylene oxide in the presence of a catalyst, said surfactantcontaining polyoxypropylene chains having an average molecular weight ofbetween about 750 and about 6750 and polyoxyethylene chains constitutingbetween about 10 and about 80 weight percent of said surfactant.
 3. ABUFFER SUBSTRATE REAGENT FORMULATION CONTAINING: A. B-GLYCEROPHOSPHORICACID DISPDIUM SALT; B. TRIS-(HYDROXYMETHYL)-AMINOMETHANE; C. SUCCINICACID; D. SODIUM LAURYL SULFATE; AND E. SAID NITROGEN-CONTAININGPOLYOXYALKYLENE NONIONIC SURFACTANT; THE METHOD COMPRISING THE STEPS OF:I. DISSOLVING EACH OF SAID REAGENT FOMRULATIONS IN SEPARATE PORTIONS OFWATER TO PRODUCE A PLURALITY OF LIQUID REAGENTS; II. PREHEATING ANALIQUOT OF PREDTERMINED VOLUME OF THE LIQUID REAGENT CONTAINING SAIDBUFFER SUBSTRATE REAGENT FORULATION FOR A PREDETERMINED PERIOD OF TIME;III. MIXING THE PREHEATED ALIQUOT OF SAID LIQUID REAGENT COMTAINING SAIDBUFFER SUBSTRATE REAGENT FORMULATION WITH A BIOLOGICAL SPECIMEN TO FORMA SPECIMEN/REAGENT TEST SYSTEM; IV. MAINTAINING THE SPECIMEN/REAGENTTEST SYSTEM AT A PREDETERMINED ELEVATED TEMPERATURE FOR A PREDETERMINEDPERIOD OF TIME; V. ADDING BOTH THE LIQUID REAGENT CONTAINING SAIDCHROMOGENIC REAGENT FORMULATION AND THE LIQUID REAGENT CONTAINING SAIDREDUCED QUENCH REAGENT FORMULATION TO SAID SYSTEM; VI. THEREAFTERMAINTAINING SAID SYSTEM AT A PREDETERMINED ELEVATED TEMPERATURE FOR AFURTHER PREDETERMINED PERIOD OF TIME; AND VII. MEASURING THE OPTICALDENSITY OF SAID SYSTEM RESULTING FROM THE INTERACTION BETWEEN SAIDREAGENTS AND SAID SPECIMEN.
 4. A reagent formulation as set forth inclaim 3 wherein said nitrogen-containing surfactant is solid and thepolyoxypropylene chains thereof have an average molecular weight of lessthan about
 4000. 5. A chromogenic reagent formulation for use inassaying a biological specimen for alkaline phosphatase comprising asolid, water-soluble substantially anhydrous, storage-stable mixturecontaining: a. sodium molybdate; b. sodium lauryl sulfate; and c. anitrogen-containing polyoxyalkylene nonionic surfactant obtained by thesequential reaction of ethylene diamine with propylene oxide andethylene oxide in the presence of a catalyst said surfactant containingpolyoxypropylene chains having an average molecular weight of betweenabout 750 and about 6750 and polyoxyethylene chains constituting betweenabout 10 and about 80 weight percent of said surfactant.
 6. A reagentformulation as set forth in claim 5 wherein said nitrogen-containingsurfactant is solid and the polyoxypropylene chains thereof have anaverage molecular weight of less than about
 4000. 7. THE METHOD OFASSAYING A BIOLOGICAL SPECIMEN FOR ALKALINE PHOSPHATASE USING APLURALITY OF SOLIDS WATER-SOLUBLE, SUBSTANTIALLY ANHYDROUS,STORAGE-STABLE REAGENT FORMULATIONS RESPECTIVELY COMPRISING:
 8. Themethod as set forth in claim 7 wherein the optical density of saidsystem is compared with the optical density of a blank processed undersuch conditions as to provide the same proportion of inorganicphosphorus as would be provided in said test system in the absence ofalkaline phosphatase.
 9. The method as set forth in claim 7 wherein saidnitrogen-containing polyoxyalkylene surfactant is solid and the averagemolecular weight of the polyoxypropylene chains thereof is less thanabout
 4000. 10. The method of preparing a solid, water-soluble,free-flowing, substantially anhydrous, storage-stable chromogenicreagent formulation for use in assaying a biological specimen foralkaline phosphatase, said method comprising the steps of:
 11. Themethod of preparing a solid, water-soluble, free-flowing, substantiallyanhydrous, storage-stable buffer substrate reagent formulation for usein assaying a biological specimen for alkaline phosphatase, said methodcomprising the steps of:
 12. The method of prepAring a solid,water-soluble, free-flowing, substantially anhydrous, storage-stablereducer quench reagent formulation for use in assaying a biologicalspecimen for alkaline phosphatase, said method comprising the steps of: